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Retargeting Oncolytic Vesicular Stomatitis Virus to Human T-Cell Lymphotropic Virus Type 1-Associated Adult T-Cell Leukemia.

Betancourt D, Ramos JC, Barber GN - J. Virol. (2015)

Bottom Line: VSV-gp160G was further noted to be highly attenuated and did not replicate efficiently in or induce significant cell death of primary CD4(+) T cells.Importantly, VSV-gp160G effectively exerted potent oncolytic activity in patient-derived ATL transplanted into NSG mice and facilitated a significant survival benefit.This effect greatly reduced neurotoxic risk associated with VSV infection while still allowing VSV to effectively target ATL cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida, USA.

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Related in: MedlinePlus

VSV-gp160G is capable of inducing apoptosis in ATL cells and depends upon CD4 and gp120 interaction. (A and B) ATL cells were infected with either VSV-XN2 or VSV-gp160G at an MOI of 0.01. Cells and supernatants were collected at 6, 12, 24, and 48 hpi. Cell death at each time point was determined by using annexin V-PI staining. Representative gating results are shown. (C) Bright-field microscopy of infected ATL cells pretreated with neutralizing antibody against CD4 (1 μg/well, MT-2, MT-4, TLO-m1) or gp120 (2 μg, VSV inoculum) for 1 h prior to infection at an MOI of 0.01. MT-2 and MT-4 cells were assessed at 24 hpi, and TLO-m1 cells were assessed at 96 hpi. (D) Cell death of infected ATL cells pretreated with neutralizing antibody was determined using annexin V-PI staining at 24 hpi. MT-2, MT-4, or TLO-m1 cells were infected with VSV-XN2 at an MOI of 0.5, 0.01, or 0.05, respectively, or infected with VSV-gp160G at an MOI of 0.1, 0.01, or 0.5, respectively. (Student t test, two tailed, equal variance; *, P < 0.01; **, P < 0.001 [panel D, untreated control versus antibody treated]).
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Figure 4: VSV-gp160G is capable of inducing apoptosis in ATL cells and depends upon CD4 and gp120 interaction. (A and B) ATL cells were infected with either VSV-XN2 or VSV-gp160G at an MOI of 0.01. Cells and supernatants were collected at 6, 12, 24, and 48 hpi. Cell death at each time point was determined by using annexin V-PI staining. Representative gating results are shown. (C) Bright-field microscopy of infected ATL cells pretreated with neutralizing antibody against CD4 (1 μg/well, MT-2, MT-4, TLO-m1) or gp120 (2 μg, VSV inoculum) for 1 h prior to infection at an MOI of 0.01. MT-2 and MT-4 cells were assessed at 24 hpi, and TLO-m1 cells were assessed at 96 hpi. (D) Cell death of infected ATL cells pretreated with neutralizing antibody was determined using annexin V-PI staining at 24 hpi. MT-2, MT-4, or TLO-m1 cells were infected with VSV-XN2 at an MOI of 0.5, 0.01, or 0.05, respectively, or infected with VSV-gp160G at an MOI of 0.1, 0.01, or 0.5, respectively. (Student t test, two tailed, equal variance; *, P < 0.01; **, P < 0.001 [panel D, untreated control versus antibody treated]).

Mentions: Next, growth kinetic assays were performed in a multicycle infection (MOI of 0.001) to analyze the replicative abilities of VSV-gp160G and VSV-XN2 in ATL cells. VSV-XN2 was able to replicate in all cell lines infected, whereas VSV-gp160G was only able to effectively replicate within the CD4+ ATL cell lines. Again, the growth kinetics of VSV-gp160G were significantly attenuated compared to VSV-XN2 (Fig. 3C). VSV-gp160G's ability to induce apoptosis was confirmed using flow cytometry and annexin V-PI staining. ATL cells were infected with either VSV-XN2 or VSV-gp160G at an MOI of 0.1, and representative gating results at 24 hpi are shown (Fig. 4A). The kinetics of VSV-mediated apoptosis in ATL cells were determined at 6, 12, 24, and 48 hpi. MT-2 cells were found to be extremely susceptible to VSV-gp160G-mediated apoptosis, even significantly more than they were to VSV-XN2. The extreme syncytia in the MT-2 cells at 48 hpi likely interfere with the flow cytometry analysis, since we expected the reading to be significantly higher considering the phenotype of the cells. MT-4 cells were highly sensitive to both constructs and TLO-m1 cells were slightly more resistant to VSV-gp160G than to VSV-XN2, with the difference being significant at 48 hpi (Fig. 4B). This analysis indicated that VSV-gp160G was able to effectively induce apoptosis in all ATL lines.


Retargeting Oncolytic Vesicular Stomatitis Virus to Human T-Cell Lymphotropic Virus Type 1-Associated Adult T-Cell Leukemia.

Betancourt D, Ramos JC, Barber GN - J. Virol. (2015)

VSV-gp160G is capable of inducing apoptosis in ATL cells and depends upon CD4 and gp120 interaction. (A and B) ATL cells were infected with either VSV-XN2 or VSV-gp160G at an MOI of 0.01. Cells and supernatants were collected at 6, 12, 24, and 48 hpi. Cell death at each time point was determined by using annexin V-PI staining. Representative gating results are shown. (C) Bright-field microscopy of infected ATL cells pretreated with neutralizing antibody against CD4 (1 μg/well, MT-2, MT-4, TLO-m1) or gp120 (2 μg, VSV inoculum) for 1 h prior to infection at an MOI of 0.01. MT-2 and MT-4 cells were assessed at 24 hpi, and TLO-m1 cells were assessed at 96 hpi. (D) Cell death of infected ATL cells pretreated with neutralizing antibody was determined using annexin V-PI staining at 24 hpi. MT-2, MT-4, or TLO-m1 cells were infected with VSV-XN2 at an MOI of 0.5, 0.01, or 0.05, respectively, or infected with VSV-gp160G at an MOI of 0.1, 0.01, or 0.5, respectively. (Student t test, two tailed, equal variance; *, P < 0.01; **, P < 0.001 [panel D, untreated control versus antibody treated]).
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Figure 4: VSV-gp160G is capable of inducing apoptosis in ATL cells and depends upon CD4 and gp120 interaction. (A and B) ATL cells were infected with either VSV-XN2 or VSV-gp160G at an MOI of 0.01. Cells and supernatants were collected at 6, 12, 24, and 48 hpi. Cell death at each time point was determined by using annexin V-PI staining. Representative gating results are shown. (C) Bright-field microscopy of infected ATL cells pretreated with neutralizing antibody against CD4 (1 μg/well, MT-2, MT-4, TLO-m1) or gp120 (2 μg, VSV inoculum) for 1 h prior to infection at an MOI of 0.01. MT-2 and MT-4 cells were assessed at 24 hpi, and TLO-m1 cells were assessed at 96 hpi. (D) Cell death of infected ATL cells pretreated with neutralizing antibody was determined using annexin V-PI staining at 24 hpi. MT-2, MT-4, or TLO-m1 cells were infected with VSV-XN2 at an MOI of 0.5, 0.01, or 0.05, respectively, or infected with VSV-gp160G at an MOI of 0.1, 0.01, or 0.5, respectively. (Student t test, two tailed, equal variance; *, P < 0.01; **, P < 0.001 [panel D, untreated control versus antibody treated]).
Mentions: Next, growth kinetic assays were performed in a multicycle infection (MOI of 0.001) to analyze the replicative abilities of VSV-gp160G and VSV-XN2 in ATL cells. VSV-XN2 was able to replicate in all cell lines infected, whereas VSV-gp160G was only able to effectively replicate within the CD4+ ATL cell lines. Again, the growth kinetics of VSV-gp160G were significantly attenuated compared to VSV-XN2 (Fig. 3C). VSV-gp160G's ability to induce apoptosis was confirmed using flow cytometry and annexin V-PI staining. ATL cells were infected with either VSV-XN2 or VSV-gp160G at an MOI of 0.1, and representative gating results at 24 hpi are shown (Fig. 4A). The kinetics of VSV-mediated apoptosis in ATL cells were determined at 6, 12, 24, and 48 hpi. MT-2 cells were found to be extremely susceptible to VSV-gp160G-mediated apoptosis, even significantly more than they were to VSV-XN2. The extreme syncytia in the MT-2 cells at 48 hpi likely interfere with the flow cytometry analysis, since we expected the reading to be significantly higher considering the phenotype of the cells. MT-4 cells were highly sensitive to both constructs and TLO-m1 cells were slightly more resistant to VSV-gp160G than to VSV-XN2, with the difference being significant at 48 hpi (Fig. 4B). This analysis indicated that VSV-gp160G was able to effectively induce apoptosis in all ATL lines.

Bottom Line: VSV-gp160G was further noted to be highly attenuated and did not replicate efficiently in or induce significant cell death of primary CD4(+) T cells.Importantly, VSV-gp160G effectively exerted potent oncolytic activity in patient-derived ATL transplanted into NSG mice and facilitated a significant survival benefit.This effect greatly reduced neurotoxic risk associated with VSV infection while still allowing VSV to effectively target ATL cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida, USA.

Show MeSH
Related in: MedlinePlus